Section 1
Suspended lipid bilayers, also known as Black lipid membranes (BLMs), are a model system used to study the function and activity of transmembrane proteins, engineered proteins, phospholipid organization, and as a key component of ion channel-based biosensors. Ion channels (IC) possess a number of desirable properties that make them useful for analytical applications, including ion selectivity, chemical or mechanical gating, inherent signal amplification, well-defined open and closed states and simple electrical readout. BLMs provide an important synthetic membrane environment to study the function and activity of ion channels and serve as key components of ion channel-functionalized analytical platforms.
Additionally, BLMs have potential in high-throughput applications, including drug screening, due to the formation of an array of BLM-IC-based biosensors. A major limitation of BLM-based platforms (i.e. ion channel-functionalized sensor platforms) is the ability to form membranes with adequate electrical, mechanical and temporal stability. BLM instability arises from the relatively weak noncovalent forces of interaction between lipid molecules in the membrane, which are insufficient to maintain the structure of BLMs under mechanical, chemical and electrical stresses. Further, the interaction forces between the lipid membrane and the underlying substrate significantly affect the temporal stability of BLMs.
The development of robust BLMs has been a major focus of research efforts. A few of the methods developed to enhance the formation and stability of BLMs include; miniaturization of aperture size, reducing the surface energy of aperture substrates, sandwiching the BLM between hydrogel layers, and chemical cross-linking by photopolymerization of reactive amphiphiles. Benz et al. pioneered the direct polymerization of lipid membranes as a method of stabilizing BLMs, and identified lipid compositions for developing synthetic ion channel-functionalized sensors (Benz, R.; Elbert, R.; Prass, W.; Ringsdorf, H. Eur. Biophys. J. 1986, 14, 83-92). Reactive chemical functionalities can be introduced in the structure of lipid amphiphiles during synthesis to allow cross-linking at the lipid head group, the middle or the distal end of the lipid tail, or via a linker attached to the lipid head group. The degree of cross-linking in polymeric membranes depends on the type of polymerizable lipid and method of polymerization used, and affects the fluidity and stability of the lipid membranes. While polymerization can significantly enhance the stability of BLMs, stiff, viscous polymeric membranes may inhibit the function of some ICs.
A number of approaches have been explored to address the challenge of creating stable membranes that retain fluidity. Schmidt and coworkers created stable long lived BLM platforms for single-channel measurements by encapsulating a pre-existing free-standing membrane within a gel polymerized around it in situ (Jeon, T. J.; Malmstadt, N.; Schmidt, J. J. J. Am. Chem. Soc. 2006, 128, 42-43). Although the lifetime of BLM was greatly enhanced, the method reduces the effective diffusion of IC into BLM by ca. 70%, thus increasing IC reconstitution time and making application of the method for sensor development impractical. BLMs have been prepared from mixtures of polymerizable and nonpolymerizable phospholipids which allowed adequate fluidity to observe normal ion channel activity. Shenoy and co-workers reported improved bilayer lifetime using a mixture of polymerizable and non-polymerizable lipids (Daly, S. M.; Heffernan, L. A.; Barger, W. R.; Shenoy, D. K. Langmuir 2005, 22, 1215-1222). They observed wide fluctuations in the lifetime of UV irradiated BLMs due to variation in the amount of reactive polymerizable lipids that formed the BLM. To address the challenge of membrane fluidity, Heitz et al demonstrated the preparation of highly stable BLMs from a mixture of polymerizable (bis-dienoyl phosphatidylcholine) and nonpolymerizable (1, 2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC)) phospholipids, a mixture that retained sufficient fluidity for reconstitution and proper function of ion channels and allowed for observation of normal IC activity (Heitz, B. A.; Jones, I. W.; Hall, H. K.; Aspinwall, C. A.; Saavedra, S. S. J. Am. Chem. Soc. 2010, 132, 7086-7093). Meier et al. used a different approach to enhance the electrical stability of free standing lipid membranes, in which BLMs were formed from a mixture of nonpolymerizable lipids and nonlipid hydrophobic monomers consisting of polymerizable styrene and divinylbenzene (Meier, W.; Graff, A.; Diederich, A.; Winterhalter, M. Phys Chem Chem Phys 2000, 2, 4559-4562). Despite the enhanced electrical stability of BLMs after UV-initiated polymerization, the longevity of the BLMs, fluidity and compatibility with ion channel reconstitution were not investigated.
Here, the present invention features a simple and cost effective method of improving the stability of BLMs from a mixture of nonpolymerizable lipids and commercially available, hydrophobic polymerizable monomers (i.e. methacrylate monomers) that partition into the lamella region of the lipid bilayer. BLMs prepared in equimolar mixture with nonlipid, hydrophobic monomers (BMA and EDGMA) were evaluated for their electrical, mechanical and physical properties before and after UV photopolymerization. The present invention shows dramatically enhanced BLM stability and maintenance of incorporated ion channel activity.
Section 2
Phospholipid membranes play key roles in the regulation of biological function by serving as a barrier between the extracellular and intracellular environments, as well as in the evaluation of physiological and pharmaceutical modulators of biological function. Additionally, phospholipid membranes provide a suitable chemical environment for expression and solubilization of transmembrane proteins. Due to the importance of phospholipid membranes in biological function, many pharmaceutical modulators interact with the macromolecular assemblies either via direct partitioning into the membrane or through interactions with transmembrane proteins.
Current drug screening assays commonly utilize intact cells to identify novel small molecule agonists and antagonists that interact directly with membranes or, more specifically, transmembrane proteins. Unfortunately, cell-based assays suffer from irreproducibility due to variability among heterogeneous cell populations, exhibit false positives and false negatives due to non-specific interactions, and are difficult to interpret due to the complexity associated with monitoring downstream effects of signal transduction. In contrast, affinity chromatography platforms that integrate phospholipid membranes present a unique capability for identifying compounds that interact directly with the membrane or with integrated membrane proteins. Additionally, membrane-functionalized affinity platforms tend to minimize non-specific interactions.
Phospholipid membrane-functionalized affinity stationary phases have been utilized in chromatography to study partitioning and binding interactions. In immobilized liposome chromatography (ILC), liposomes are retained on a support matrix through steric, hydrophobic, covalent, avidin-biotin, or other types of specific or non-specific interactions. ILC has been primarily used to study small molecule partitioning through lipid membranes and interactions between peptides and phospholipids. Various membrane proteins have been immobilized in ILC stationary phases and used to study ligand binding; however, the liposomes are formed by non-covalent interactions, which are inherently unstable. Thus, the stationary phases have limited lifetimes, as well as reduced pressure stability, requiring low flow rates that reduce separation efficiency. Furthermore, ILC phases lack the chemical and mechanical stability to withstand variations in solution conditions (e.g. small fractions of organic solvents, varying ionic strength, etc.) and physical and mechanical insults (air bubbles, shear forces, etc.), decreasing the reproducibility of the columns and limiting their utility.
Immobilized artificial membranes (IAMs) provide an alternative chromatographic stationary phase that exhibits greater stability and reproducibility than ILCs. IAMs are prepared by covalent attachment of lipid tails to an underlying support, resulting in formation of a lipid monolayer on the particle surface. IAMs have been used to study partitioning and interactions between small molecules and phospholipids. Additionally, nicotinic acetylcholine receptors, μ and κ opioid receptors, and other membrane proteins were separately immobilized in IAMs and packed into columns. Using frontal chromatography, dissociation constants for the various membrane proteins were calculated against a series of small molecules, revealing similar trends to binding constants calculated using cell-based assays. However, there were quantitative differences, likely due to the altered conformation of membrane proteins upon interaction with the underlying silica support and the truncated lipid membrane.
Affinity chromatographic matrices for analyzing membrane proteins and molecules that interact with them would benefit from the presence of a more stable lipid structure that more accurately represents a lipid bilayer to allow incorporation of a larger number of membrane proteins, while maintaining their native conformations. Bilayer stability can be increased by a number of methods, such as incorporating cholesterol, adsorbing a protective overlayer, membrane tethering, and polymerizing phospholipid monomers. Of these methods, direct polymerization of phospholipid monomers yields the most stable structures.
Polymerizable phospholipids have been used in various analytical platforms to form stable phospholipid bilayers. As a non-limiting example, planar supported lipid bilayers (PSLBs) prepared by polymerizing bis-SorbPC (1,2-bis[10-(2′,4′-hexadieoyloxy)decanoyl]-sn-glycero-2-phosphocholine) have exhibited stability against surfactants, organics, and exposure to high vacuum. Polymerized bis-SorbPC (poly(bis-SorbPC)) membranes have also been utilized in capillary zone electrophoresis as stable surface coatings for reducing the electro-osmotic flow and minimizing non-specific adsorption of proteins. These polymerized surface coatings were stable to surfactant solutions, shear forces, applied electric fields, and dry storage. Additionally, Rhodopsin, a transmembrane protein, was incorporated into PSLBs prepared from poly(bis-SorbPC) and retained its activity in the stabilized bilayer. When combined, these data support further investigation of polymeric lipid bilayers for enhancing stability of phospholipid-based stationary phases and the utility of the resulting polymeric lipid stationary phases for chromatographic separations.
Though polymeric-lipid coatings have been used to minimize non-specific adsorption of proteins in a range of materials, to the knowledge of the inventors, polymeric-lipid membranes have not yet been utilized as a stationary phase material in packed columns. The present invention features a method of preparing poly(bis-SorbPC) coatings onto silica particles that were subsequently packed into capillary LC columns. The chemical, physical, and temporal stability of the polymerized-phospholipid bilayers were assessed and their utility as a lipid-based stationary phase for liquid chromatography is demonstrated.
Polymer lipid membranes can be prepared by photochemical or redox initiated polymerization of synthetic, polymerizable lipid. The resulting polymerized stationary phases exhibited enhanced stability compared to particles coated with non-polymerizable lipid bilayers when exposed to chemical and physical assaults over a period of time. However, a drawback to redox polymerization is that initiation and progression of the polymerization using conventional redox mixtures proceed only under acidic conditions, which poses a problem for proteins incorporated into the lipid bilayer membrane. For example, transmembrane proteins often get denatured, and activities of these proteins can diminish or disappear after redox polymerization with current redox mixtures. Hence there is a need for redox mixtures that provide milder conditions when used in the polymerization of lipids. The present invention features a redox mixture that allows for polymerization methods to proceed under neutral pH conditions while preserving the proteins incorporated into the polymerized lipid membrane.
Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.